Antibiotics are among the most important and widely used medicines. Their extensive and unrestricted use has, however, increased the rate at which pathogenic microbes develop resistant phenotypes. Traditional approaches have focused on the development of antibacterials, which cannot provide enough compounds in the pipeline to account for resistance emergence. There is now a dire need for alternative strategies. We have recently developed an innovative and systematic strategy to develop resistance-modifying agents (RMAs) that re-sensitize resistant bacteria to old antibacterials. RMAs are intriguing because they can extend the useful life span of the current, cost-effective antibiotics with well-studied toxicity profiles. In addition, they do not target essential genes, and thus pose no or litle selective pressure on bacteria. The chance of developing resistance to this strategy is smaller than for conventional antibacterials. However, the only class of RMAs that have been proven clinically useful is ?-lactamase inhibitors. Using a bio-inspired approach, we recently discovered two new scaffolds of ?-lactam-potentiating reagents in methicillin-resistant Staphylococcus aureus (MRSA) that are not ?-lactamase inhibitors. The objective of this application is to further optimize these two lead RMAs and demonstrate their efficacy in in vivo mouse models. The rationale is that the RMAs developed here may serve as drug candidates with novel mechanisms of action, which will be ready for investigational new drug (IND) enabling studies and subsequent clinical development. Specifically, we plan to: in R21 phase, Aim 1. optimize the potency, selectivity, mammalian toxicity, and drug-like properties of our recently discovered RMAs; Aim 2. optimize the pharmacokinetic properties of our lead RMAs; and in R33 phase, Aim 3. characterize the effectiveness of lead RMAs in in vivo mouse models, improve in vivo efficacy and reduce potential liabilities; Aim 4. identify the molecular targets of lead RMAs. As resistant bacteria often share conserved resistance mechanisms, the in vivo active RMAs developed form this work may also be investigated for use against infections from other resistant bacteria. The molecular targets identified here will also inspire the discovery of additional RMAs using targeted approaches. In addition, successful accomplishment of the proposed studies will also validate our approach and inspire the development of novel RMAs to extend the lifespan of other antibacterials currently used in the clinic.